This invention relates generally to the use of recombinant DNA methods for genetically altering plants. In particular, it relates to the modulation of the levels of sugars in plants using recombinant DNA.
The growth and development of plants requires the products of the metabolic pathways that provide carbon. Two critical products of these pathways are sucrose and starch. UDP-glucose pyrophosphorylase (UGPase) (FIG. 1, reaction 1) is a key enzyme in the biosynthesis of both carbohydrates. Early in development the leaf is a sink, and the triose-phosphates that are the product of CO.sub.2 fixation in the chloroplast are converted through a series of steps to the six carbon molecule glucose-1-phosphate. Through the action of ADP-glucose pyrophosphorylase (AGPase) (FIG. 1, reaction 9), this molecule is converted to ADP-glucose which is the primary substrate for starch biosynthesis. As the leaf matures it becomes a source of carbon for the growth of other tissues, and the metabolism switches to sucrose biosynthesis. At this point, the triose phosphates are transported to the cytoplasm and are converted to glucose-1-phosphate by the same reactions that occur in the chloroplast. The glucose-1-phosphate is then converted by UGPase to UDP-glucose which is a substrate for sucrose biosynthesis (FIG. 1, reactions 2 and 3). Xu et al. Plant Physiol. 90:635-642 (1989).
UGPase activity is also associated with non-photosynthetic sink tissues such as the potato tuber. When sucrose is delivered to sink tissues, it is cleaved by sucrose synthase (SS) (FIG. 1, reaction 5) to UDP-glucose and fructose. UGPase then converts the UDP-glucose to glucose-1-phosphate. At this point, the hexose phosphate enters the amyloplast and serves as substrate for AGPase, and subsequently starch synthesis. Under stress conditions, starch is degraded and glucose-1-phosphate is released from the amyloplast and can then enter the same cytoplasmic sucrose biosynthetic pathway involving UGPase described above.
Regulation of the interconversion of starch and sugars is of significant commercial interest. For instance, starch breakdown in the tuber is an important consideration to the potato industry. During cold storage, the reducing sugars glucose and fructose accumulate after starch degradation. The hexose accumulation begins when glucose-1-phosphate is released during starch degradation (FIG. 1, reaction 13). As in the leaf cytoplasm, the glucose-1-phosphate is converted to sucrose by UGPase, sucrose phosphate synthase (SPS), and sucrose-6-phosphate phosphatase. Hexoses are then formed when invertase cleaves sucrose into its hexose sugar components (FIG. 1, reaction 4).
The accumulation of hexose sugars leads to a darkening of chips or fries during cooking as a result of a non-enzymatic Maillard reaction which involves a condensation between the free sugar aldehydes and the amine groups of amino acids. Thus, methods of controlling the expression of the genes in these metabolic pathways could lead to improved commercial varieties of potatoes and other plants.
One potential method of controlling plant gene expression is the use of ribozymes. Only circumstantial evidence of the activity of ribozymes in plants has been reported in literature. The self-splicing of Chlamydomonas reinhardtii chloroplast ribosomal RNA was the first evidence of autocatalytic RNA activity in plant kingdom. Durrenberger et al., EMBO J. 10:3495-3501 (1991). Recently a specific activity of a hammerhead-type ribozyme has been shown in Nicotiana tabacum protoplasts (Steinecke et al., 1992). No report of the ability of ribozymes to produce a phenotypic change in transgenic plants has been shown, however.
A need exists to identify economical methods for controlling metabolic pathways associated with starch synthesis. In particular, new methods of inhibiting gene expression (e.g., by ribozymes) in transgenic plants would provide new techniques for modifying a number of plant traits.